The Metabolomics of Nitric Oxide and Reactive Nitrogen Species in Immune Editing Tumor Milieu: Influence of Nitric Oxide-Modulating Therapies Ashok R

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ReviewResearch Article Article OpenOpen Access Access The Metabolomics of Nitric Oxide and Reactive Nitrogen Species in Immune Editing Tumor Milieu: Influence of Nitric Oxide-Modulating Therapies Ashok R. Amin* Department of Biochemical Engineering, Virginia Tech, Virginia College of Osteopathic Medicine. Rheumatrix Inc., Blacksburg, Virginia 24061, USA

Abstract Nitric oxide (NO), reactive nitrogen and oxygen species, hypoxia, L-arginine metabolites and peroxynitrite are contributing agents during various stages of carcinogenesis. Among these, NO contributes to various functions in the tumor microenvironment such as augmenting immune suppression, maintaining anergy to tumor associated antigens, stimulating angiogenesis, enhancing tumor growth and invasiveness, and modulating autophagy. Most cells in the tumor milieu either release NO and/or have altered physiological functions due to the influence of NO. Cancer cells evolve into a metabolic state to tolerate and reduce reactive nitrogen and oxygen species to elude oxidative damage. These cancer cells in the tumor microenvironment inhibit cellular infiltration of effector immune cells into the cancer milieu. Increased levels of CCAAT-enhancer-binding proteins such as C/EBP α and β are required for inducible Nitric Oxide Synthase (iNOS) gene expression and other transcripts for sustaining an immunosuppressive environment in growing tumors. The immunoediting property of reactive nitrogen and oxygen-modified metabolites change the functions of: intratumoral chemokines, T cell receptors, antigen specific tumor infiltrating lymphocytes (TILs), cytotoxic T lymphocyte, myeloid derived suppressive cells (MDSCs), gene expression, tumor suppressor genes, and Interferon responses which together countersignal tumor immunity. Recent studies show that reactive nitrogen species that promote tumor-mediated immune evasion can be reversed by gene therapy, immunotherapy, and chemotherapy by targeting or co-targeting excessive nitric oxide accumulation.

Keywords: Nitric oxide; Free radicals; Cancer; Metabolomics; systems [6]. It is highly reactive and diffuses freely across cell Immunotherapy; Chemotherapy membranes. This property of NO makes it an ideal transient paracrine and autocrine signaling molecule in biological systems. Five chemical Abbreviations: Nitric oxide: NO; Nitric oxide synthase: NOS; processes occur in the biological milieu upon exposure to NO: nitration Inducible Nitric Oxide Synthase: iNOS; Peroxynitrite: (ONOO-); and nitrosation, nitrosylation, oxidation and interactions with other Superoxide: (O2−); Reactive nitrogen species: RNS; Reactive oxygen free radicals, e.g., H2O2 [7]. All the five effects of NO have been reported species: ROS; Tumor infiltrating lymphocytes: TILs; Myeloid derived to be involved in generation of modified metabolites in various cell suppressive cells: MDSCs; L-arginine: L-Arg; L-Citrulline: L-Cit; signaling processes [3,8,9] (Figure1). Cytotoxic T cells: CTL; T regulatory cells: Tregs; Immature myeloid cells: IMCs; L- Arginine: L-Arg; Cytoplasmic arginase: Arginase 1; Nitric oxide is biosynthesized by three isoforms of nitric oxide Mitochondrial arginase: Arginase2; Non-steroidal anti-inflammatory synthases (NOSs). Endothelial NOS (eNOS), neuronal NOS (nNOS) drugs: NSAIDS; Superoxide dismutase: SOD; Pentose Phosphate and, inducible NOS (iNOS) are all involved in immune responses Pathway: PPP; Hypoxia-inducible factor-1α protein: HIF-1α [3,7-9]. All the isoforms of NOS utilize L-arginine (L-Arg), oxygen, tetrahydrobiopterin and NADPH to generate NO and L-citrulline Cancer Immunoediting Come of Age (L-Cit) [6,7]. Among these isoforms, iNOS exhibits high NO output and has been reported in various cell types, which include but not limited The development in immunology in the last two decades has to M2 macrophages, MDSCs, dendritic cells, NK cells, tumor cells, given us a different level of appreciation of the immune system and endothelial cells, neuronal cells, and neutrophils which are involved the dual role it plays in cancer. The immune surveillance system in inflammation and cancer [10-16]. Cancer cells have a tendency suppresses tumor formation by destroying cancer cells or obstructing to disregard oxygen availability, electing for less efficient anaerobic their outgrowth. The development of some cancers might be seen as pathways of generating energy such as the pentose phosphate pathway a failure of immune surveillance and the ability of tumors to thwart and avoiding glycolysis [17]. The astuteness behind this is to manage the development of effective immune responses against their antigens. the reactive oxygen species and or oxidative damage to itself in the This resistance of tumors against the immune response is facilitated tumor microenvironment. This process is specific for cancer cells, as by immunoediting of immune signals induced by the tumors and their metabolites during the course of carcinogenesis [1,2]. One of the components of the immune system that initiates the mechanism of *Corresponding author: Ashok R. Amin, Rheumatrix Inc., Blacksburg, Virginia cancer immunoediting is inflammation [3-5]. Some of the tools (such 24060, USA, Tel: +908-416-5739; E-mail: [email protected] as nitric oxide and reactive nitrogen and or oxygen species) utilized by Received January 13, 2012; Accepted July 06, 2012; Published July 09, 2012 the immune system during infections and host defense also function to Citation: Amin AR (2012) The Metabolomics of Nitric Oxide and Reactive Nitrogen promote immune suppression in the tumor microenvironment. Species in Immune Editing Tumor Milieu: Influence of Nitric Oxide-Modulating Therapies. J Drug Metab Toxicol S8:002. doi:10.4172/2157-7609.S8-002

Nitric oxide in cancer, a compelling relationship in tumor Copyright: © 2012 Amin AR, et al. This is an open-access article distributed under progression the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and Nitric oxide is a gaseous signaling molecule in various biological source are credited.

Mechanisms in Free radical J Drug Metab Toxicol Metabolomics ISSN: 2157-7609 JDMT, an open access journal Citation: Amin AR (2012) The Metabolomics of Nitric Oxide and Reactive Nitrogen Species in Immune Editing Tumor Milieu: Influence of Nitric Oxide- Modulating Therapies. J Drug Metab Toxicol S8:002. doi:10.4172/2157-7609.S8-002

Page 2 of 7 it may damage other metabolic active cells such as liver and immune cells. Thus “cancer cells make sacrifices for survival” [17].   

Reduce RNS & Target specific Increase RNS & Low level of NO is essential for immune functions whereas ROS burden by RNS & ROS- ROS toxicity by chemotherapy generating cells Gene Therapy high levels of NO are associated with immune suppression [18]. or combinational or antigen therapy Several studies using NOS-/- mice and/or NOS inhibitors have demonstrated that NO mostly promotes tumor progression as reviewed by Fukumura et al. [19]. Cells of the tumor milieu (Figure 2) adapt to metabolic changes to limit the toxicity of free radicals, and acquire tumor immunity, resistance to apoptosis or other forms   of cell death [7,17,20,21]. Thus NO is not only tightly regulated in the    tumor environment, but it undergoes constant change and exhibits    heterogenerous effects depending on the type of tumor, concentration of NO, NO’s ability to interact with other free radicals, proteins, metal ions and genetic background of its target [18]. The iNOS activity can  change during tumor progression. For example, in colon cancers, iNOS Figure 2: Strategies for modulating nitric oxide, peroxynitrite, and superoxide activity is at the highest in adenomas. This iNOS activity decreases with in tumor milieu for therapy: The figure shows the various targets in the tumor advancing tumor-stages and was found to be minimum in metastatic milieu for therapeutic intervention. Reactive nitrogen and oxygen species and peroxynitrite within the tumor milieu are necessary and collective contributors tumors [19]. We and others have reported the pleiotropic role of NO in of tumor-mediated immune evasion.(3, 19). They may function independently normal and pathophysiological conditions [22] including cancers [23]. or collaboratively depending on the biochemical event. The cancer cells have In the present review, we will focus on the role of NO, NO producing evolved into a metabolic state to not only tolerate, but reduce reactive free radicals and oxidative damage. This metabolic state is regulated by various cells, the modification of metabolites by free radicals and their impact biochemical pathways {e.g. pentose phosphate pathways (PPP)}, enzymes on other cells involved in tumor immunity, immunotherapy, and {e.g. superoxide dismutase (SOD)} and other factors {such as hypoxia-induc- chemotherapy. ible factors (HIFs)} in the tumor milieu [17,56,57,71]. Several strategies have shown promising results in reversing immunosuppression by disrupting the Free radical releasing cells in tumor milieu roles of reactive nitrogen and oxygen species in tumors in animal studies and in man [56,57] These approaches include immunotherapy, gene therapy and The tumor milieu is a complex system of many cells [24]. They all chemotherapy [19,56,57,71]. Gene therapy targets the basic redox balancing processes. This approach is cancer-cell specific and it avoids collateral damage. Various chemotherapies and immune therapies reduce the levels of reactive nitrogen and/or oxygen species as secondary targets. Inhibitors of reactive Impairs nitrogen and or oxygen species have been used during Adoptive Cell Therapy T cell or in vivo for preconditioning tumor microenvironment with other combinational functions therapies [19,46]. Another approach is by increasing the levels of endogenous  nitric acid in tumors or introducing exogenous NO which sensitizes tumors to   radiation therapy [19].     Nitric Oxide participate in tumor progression, immunity, and immune suppression Peroxynitrite [5,25,26]. These cells include endothelial cells, pericytes, fibroblasts of Superoxide various phenotypes, neutrophils and other granulocytes (eosinophils     and basophils), cytotoxic T cells (CTL), mast cells, CD4 and CD8 T   cells, B cells, natural killer (NK) lymphocytes, antigen-presenting cells   such as macrophages, and dendritic cells [5,25,26]. Among these are  cells with specialized functions such as T regulatory cells (Tregs) and  MDSC [2,10,14,27]. The phenotypes of tumor-associated macrophages (TAMs) are distinct from normal macrophages [11]. Tumor-associated Figure 1: Nitric oxide, peroxynitrite, and superoxide promote immunosuppres- macrophages exhibit pro-tumoural functions and participate in sion in tumor milieu: Most tumors show increased levels of free radicals which suppression of adaptive immunity [11]. They also exhibit functional include reactive nitrogen and or oxygen species; however, cancer cells disre- plasticity and also show reversible adaptation to changing environments gard the availability of oxygen and choose a less efficient path for generating [16,28].The TAMs, MDSCs, dendritic cells, NK cells, tumor cells, energy. Cancer cells operate via the pentose phosphate pathway (PPP) for their energy which generates fewer free radicals as compared to the glycolysis. neutrophils, eosinophils, basophils, and endothelial cells have been The aim is to prevent the buildup of reactive nitrogen species and reactive oxy- reported to release NO and free radicals, which influence physiological gen species and oxidative damage. In the process, cancer cells show increased functions in the tumor microenvironment [10,11,19,21,29-32]. levels of superoxide dismutase (SOD) activity and hypoxia-inducible protein factors (HIFs). HIF-1α is a transcription factor which induces transcription of The hijacking of myeloid-derived suppressor cells (MDSCs) several genes in hypoxia and low oxygen tension for cell survival. SODs are responsible for eliminating free radicals. The reactive nitrogen and or oxygen spe- by tumors cies support conditions of immunosuppression in the tumor microenvironment. Some of the mechanisms include modification of metabolites of transformed The immune system is structured to protect itself from excessive cells and other cells in the vicinity such as impairing the activities of T and NK immune stimulation induced by cancer and/or self-antigens released cells that are involved in tumor regression. Reactive nitrogen species modify by trauma. In the process, a complex network of soluble factors chemokine that are required for signals that facilitate the entry of effectors cells into the tumor but allow myeloid cells which promote immunosuppression. Nitric stimulates the production of immature myeloid cells (IMC) from the oxide modifies various signal transduction molecules within the cells that alter bone marrow and into the blood circulation [33,34]. the functions of various cells with respect to autophagy and apoptosis. It also modulates oncogenes like p53 which regulate apoptosis [7]. Reactive nitrogen Increased amounts of IMCs are also associated with a state of and or oxygen species influence the changes in the cell-surface markers and immune suppression, dysfunctional dendritic cells, and T cells [35]. modify cell phenotypes involved in carcinogenesis. These IMCs can also generate a separate lineage of cells with distinct

Page 3 of 7 immunoregulatory properties, which include MDSCs. While MDSCs spleen where splenic MDSCs also down-regulated of CD3 zeta chain have been reported to have a role in wound healing and tissue repair, expression in antigen-stimulated CD4+but not CD8+ T cells. Another tumors have learned to ‘harness’, these characteristics of MDSCs for mechanism where L-Arg metabolism augments T cell suppression is antitumor immunity and tolerance, promote tumor development, and reported in individuals with prostate cancer [35]. Increased expression metastasis [16]. Myeloid derived suppressive cells are characterized of arginase 2 and iNOS by cancer cells inhibited CD8+ tumor- by increased levels of arginase activity, reactive oxygen species and infiltrating lymphocytes. These+ CD8 tumor-infiltrating lymphocytes NO. In mice, their phenotype is CD11b+Gr1+ and in humans, it is did not show any changes in the expression of CD3 zeta chain or other CD11b+CD14-CD33+ or Lin-HLA-DR- CD33+ [11,16,36]. The human significant deficiencies in the TCR signaling mechanisms [29,41]. The cells do not express the Gr1 homologue. The human population third possible mechanism to inhibit T cell proliferation in tumors of MDSCs in blood shows the CD15+ marker [16]. Myeloid derived milieu is mediated by neutrophils, which showed increased levels of suppressive cells do not exhibit an immunosuppressive phenotype in arginase 1 and depletion of extracellular L-Arg [15].The mechanism the bone marrow, but that may change in the lymphoid organs and of immune suppression by neutrophils was associatedwith release of cancer due to the inflammatory environment, growth factors, cytokines azurophil granules. and chemokines. In the tumor milieu, MDSC differentiates into a The upregulation of NO in several human cancers contribute tumor associated macrophages like phenotype and retain their ability to neoangiogenesis, tumor metastasis, tumor-related immune to generate NO and free radicals [16]. Kilinc et al. (2011) 1suggested suppression, and autophagy by modification of signal transduction that iNOS+- myeloid cells in tumors have multiple effector functions. pathways [18,19].Unlike the increased activity of arginase, which They are also phenotypically similar to TAMs. These cells couldbe leads to paralysis of T cell functions due to depletion of the CD3 zeta immune stained for iNOS expression but could be separated into two chain, increased levels of NO inhibit JAK-1 and -3, Erk, Akt, STAT5 populations (P1) CD11bhigh, Gr1low/- F4/80+ cells and (P2) CD11b low/- phosphorylation, and blocks IL-2 signaling In T cells [12,19]. Nitric , Gr1 low/- F4/80. These populations of cells were also observed in the oxide modulates autophagy by S-nitrosylation of JNK1 and IKKβ. bone marrow and blood and their accumulation was dependent on In the process, inhibition of JNK1 decreases Bcl-2 phosphorylation, tumor growth. Furthermore, In vitro experiments showed that these and blocks the formation of the hVps34/Beclin 1 complex and iNOS+ myeloid cells in the tumors could inhibit proliferation of CD8+ T related signaling pathway. Nitric Oxide induces inhibition of IKKβ, cells and induce apoptosis which could be reversed by iNOS inhibitors decreases AMPK phosphorylation and activates TSC2 and mTORC1 [1]. These intra-tumoral MDSCs acquire strong immunosuppressive [42]. Overexpression of all isoforms of NOS impairs autophagosome activity as described below by utilizing NO and other free radicals as formation through the JNK1–Bcl-2 pathway and in the process one of the arsenals for their effector functions. facilitates tumor growth [42]. S-nitrosylation of caspase 3 (at L-Arginine metabolism, reactive nitrogen species and Cys163) results in decreased apoptosis of cells, where S-nitrosylation peroxynitrites in immunoediting of immune signals of p21Ras (at Cys118) results in activation of Ras and increased proliferation and migration of endothelial cells [19,43,44]. Increased L-Arginine is a substrate to three enzymes involved in the regulation levels of NO is associated with G:C to A:T mutation in oncogene of free radicals. Cytoplasmic arginase (arginase 1) and mitochondrial p53 at 5-methylcytosine site in breast, brain and stomach cancer [3]. arginase (arginase 2) hydrolyze L-Arg to urea and ornithine. Ornithine Increased levels of nitric oxide in MDSCs correlated with nitration of is converted to polyamines by ornithine decarboxylase [16,19]. iNOS STAT1 (Ni-STAT1) and inhibition of phosphorylation of Try 701. The oxidizes L-Arg to L-Cit and releases NO [37]. An increased level of Ni-STAT1 coincided with reduced IFN-response in immune cells in arginase and iNOS activity has been documented in patients with animal models of adenocarcinomas which could be reversed in iNOS melanomas, gliomas, sarcomas, prostate, breast, colon, and lung cancer -/- mice [19]. [16,19,38]. Previous studies have proposed that the increased arginase enzymatic activity in tumors is required to sustain the high demand Post-translational modification of DNA bases, amino acids, of polyamines necessary for tumor growth [39]. Furthermore, specific and chemokines have been reported in tumor micro environments oncogenes and tumor-suppressor genes have also been reported to with increased levels of reactive nitrogen species and peroxynitrite regulate polyamine metabolism [39]. [3,45]. The production of reactive nitrogen species induce nitration of Chemokine (C-C motif) ligand 2- (CCL2) leading to formation of Tumor infiltrating lymphocytes cells (TILs) migrate from the N-CCL2, which deters T cell infiltration into the tumor. Although blood stream into the tumor. In most solid tumors, TILs are unable N-CCL2 trapped tumor-specific T cells in the stroma that surrounded to kill autologous tumors and are predominantly in an anergic state cancer cells, N-CCL2 attracted myeloid cells into the tumor [46-48]. [16,40]. Increased arginase activity in tumor infiltrating macrophages The action of N-CCL2 was reversible by preconditioning of the tumor diminished synthesis of the CD3 zeta chain and antigen-specific T cell milieu with drugs (with peroxynitrite–scavenging activity) that block responses [16,40]. Macrophages stimulated with IL-4 plus IL-13 up- CCL2 nitration, which also facilitated CTL invasion of the tumor. regulated arginase1 and amino acid transporter 2B, which triggered a Furthermore, NO induces changes in cellular phenotypes that promote rapid reduction in the levels of L-Arg in the extracellular compartment. immune suppression. For example, NO facilitated transformation of This coincided with decreased expression of CD3 zeta chain inT CD4+CD25+ Foxp3+ regulatory T cells from CD4+CD25− T cells via lymphocytes and reduced proliferation of T lymphocytes [29]. Addition p53 and IL-2 [16,19]. Recent studies have also shown a close association of excess L-Arg or competitive inhibitors of arginase 1 reversed the between NO and NK cell-mediated target-cell-killing. For instance, expression of CD3 zeta chain and recovered the proliferation of T OX40 NK cell’s cytotoxic activity is dependent on NO synthesis lymphocytes. In contrast, inhibitors of iNOS inhibitors or arginase 2 and inhibitors of NO synthesis impair NK cell-mediated target cell failed to significantly reduce the extracellular levels of L-Arg or restore killing [49]. Several studies have shown that Th1 and Th2 cytokines CD3 zeta chain function [29]. The loss of CD3 zeta chain was more competitively regulate arginase and iNOS within the intracellular significant for inhibition of CD4+ T cell than of CD8+ T cell function. biochemical pathways, negative feed-back loops, and competition for This cancer related immunosuppressive activity is also observed in the the same substrate [16]. The dual activation of arginase and iNOS in

Page 4 of 7 myeloid cells unleash a powerful inhibitory signals preventing antigen- conditioned- MDSCs {by their iNOS and NADPH oxidases} can specific T lymphocytes to penetrate the tumor and eventually leading modify tyrosine residues on the T cell receptors and CD8 receptors, to apoptotic death of these antigen-specific T lymphocytes [35]. When resulting in a decreased recognition of peptide–MHC complexes [58]. iNOS and arginase 1 are induced together, peroxynitrite generated Since modifications of CCR2 chemokines in the tumor environment by iNOS under conditions of limiting L-arginine, causes activated T can block the entry of effector cells into the tumor environments, lymphocytes to undergo apoptosis [35]. Thus iNOS and arginase 1 therapeutic intervention to avert the formation of N-CCR2 expression may act separately or synergistically in vivo in cancer milieu. Cancer may be one approach for effective active immunotherapy [46]. cells depend on intricate antioxidative reactions which supply large Several immunization protocols have shifted the function of NO in amounts of reducing equivalents that neutralize reactive oxygen species [48]. The isoforms of Pyruvate Kinase (PKMs) regulate anti- immunotherapy. For example, Ribavirin (RBV) has been reported to oxidative metabolism in cancer cells. Lung cancer cells which show change an immune response from Th2 toward a Th1 cytokine profile oxidation of PKM2 (on Cys 358) exhibited decreased PKM2 activity and also inhibit tetrahydrobiopterin synthesis: an essential cofactor shifting glucose-6-phosphate to the pentose phosphate pathway [17]. for the generation of NO by NOS. Ribavirin decreases NO production The activation of this pathway in cancer cells is essential for limiting and can be useful for sensitizing the treatment of melanomas, which reactive oxygen species accumulation, reducing oxidative stress, and are known to overexpress NOS [59]. Similarly, Kahn et al. [60] have tumor growth [17]. shown that adjuvant based immunotherapy required functional iNOS synthesis before procuring immunoprotective effects of CCAAT-enhancer-binding proteins (C/EBP) are common immunotherapy. These adjuvant immunized mice showed high regulators for iNOS gene expression, tumor induced tolerance, and expression of iNOS expression in spleen and lymph nodes [60]. Weiss immune suppression [50]. The regulation of MDSCs is influenced et al. [61] showed that iNOS expressed in the macrophages controlled by the cytokines such as GM-CSF, G-CSF and IL-6, which are also metastasis of renal carcinomas to the lung during IL-2/anti-CD40 dependent on CCAAT-enhancer-binding proteins. These cytokines immunotherapy [62]. These outcomes highlight the feasibility of using promote the production of MDSCs from precursors present in bone NO-modulating agents in combination-immunotherapy to build new marrow (BM). The BM-MDSCs were dependent on the transcription strategies for cancer therapy. factor C/EBPβ for some of their effector functions [51]. Adoptive transfer of tumor antigen-specific CD8+ T cells in mice devoid of C/ Tumor cells can be targeted by increasing NO toxicity in the EBPβ in myeloid cells gave rise to promising therapy in established tumor microenvironment. Several studies have shown that targeted tumors [49-51]. These observations suggested that C/EBPβ is not only overloading of reactive oxygen and nitrogen species render cancer cells a key regulator of iNOS expression [52,53], but also regulates several susceptible to oxidative damage by neutralizing the protective shield other transcripts (such as GM-CSF, G-CSF and IL-6) that foster the of the Warburg’s effects [17]. For example, this can be achieved by immunosuppressive milieu structured by growing cancers [49-53]. transfection of iNOS gene which inhibited growth of several cancers [19]. Similarly, retroviral vector-expression iNOS and an anti-carcino Challenges and promises of targeting NO, reactive nitrogen embryonic antigen (CEA) antibody chain targeted tumor cells and and oxygen species -modified metabolites for immunotherapy and induced apoptosis of malignant cells [62]. Injection of NO-releasing chemotherapy. agents such as iNOS-expressing-microencapsulated cells amplified Small molecules and biologics are being developed to create the levels of FAS and FASL protein in tumors and inhibited growth isozyme-specific inhibitors for both nitric oxide synthases and of colon and ovarian cancers [19]. These approaches also improved arginases [19,54,55]. It should be noted that inhibitors of arginase 1 may vascular density and radio-sensitivity in human colorectal cancer. interfere in the final cytosolic step of the urea cycle and the clearance Nitric oxide–producing hypoxic cells are sensitive to radiation due of nitrogenous waste in the liver [19,39]. Thus arginase inhibitors may to impaired DNA damage repair mechanisms [63]. iNOS expression cause hyperammonemia. Selective iNOS antagonists such as N-nitro- in colon cancer potentiates radiation treatment [19]. Activation of L-arginine methyl ester have also been reported to inhibit arginase eNOS by low dose radiation increased blood flow into tumors and also activity [12]. Another iNOS-selective inhibitor (1400W), inhibited increased radiation sensitivity in fibrosarcomas, liver, and lung cancers growth of iNOS expressing tumors [19]. [19]. Co-activation of arginase and iNOS within the same environment Non-steroidal anti-inflammatory drugs (NSAIDS) have been can generate the production of several types of reactive nitrogen and reported to play a protective role in various tumors [64]. We and oxygen species. For example, solid tumor environments which are in others have previously reported the ability of Aspirin to inhibit iNOS a hypoxic state with increased arginase 1 activity deplete L-arginine at clinically relevant concentrations by acetylating NOS [65]. NSAIDS concentrations. The uncoupling reaction by the NOS reductase in combination with NO donors seem to be more effective therapeutic domain at low L-arginine concentrations release superoxides (O2- agents. Nitro-aspirin (NO-ASA) inhibits both arginase1 and iNOS ) [12], which reacts immediately with residual NO, leading to the activities in lymphoid organs, spleen, and tumor-related myeloid formation of peroxinitrite [3,12]. Superoxide dismutases (SODs) cells [66,67]. Administration of a mixture of NO-ASA in colon cancer which are unregulated in cancer cells and MDSCs are vital enzymes models showed additive effects and strong synergism with increased that eliminate superoxide radical (O2-) and therefore, protect cancer reduction in tumor growth than single-drug treatments [68]. Nitro- cells from injury induced by free radicals [56]. Several reports in the aspirin may sensitize cancer cells for the effects of other antitumor literate describe SOD as a potential target for immunotherapy [57]. drugs. Bronte et al., designed a NO donor designated as AT38. AT38 Recent studies suggest that therapeutic failure of cancer correlated with promoted a massive T cell infiltration into the tumor milieu in prostate high contents of free radicals and peroxynitrite in tumors [19,46,47]. cancer to augment tumor regression by immunotherapy [46]. Michaud Peroxynitrite has been described to react with several amino acids by et al. showed that reducing the activity of two genetic determinants: directly modifying tryptophan, methionine, cysteine, phenylalanine, ATG5 and ATG7 (which regulate autophagy) by chemotherapy- histidine and typtophan [45]. Peroxynitrite that is generated by tumor induced autophagy in mouse tumor cells [69]. A preclinical study with

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Hydroxychloroquine (HCQ) which also regulates autophagy (and immunization or therapeutic timetable and suppressive networks iNOS expression) increases the efficacy of chemotherapy. Treatment present in a particular tumor. The complete inhibition of iNOS activity of patients with metastatic melanomas in Phase I studies with and or other free radicals (which are also required for housekeeping Temsirolimus plus HCQ showed stabilization of tumors in 73% of the activity) may not be necessary to reverse immune tolerance in cancer. patients as compared to none with Temsirolimus alone [70]. These modulators may be effective in combination with other therapies: For example, Chemically Modified Tetracyclines with reduced Hypoxia is known to induce iNOS gene expression and up-regulate anti-microbial activity such as ORACEATM whose pharmacological peroxynitrite production during malignant transformation [71]. HIF1α properties include decreasing free radicals, inflammation, and nitric was shown to drive the sequence of events leading to upregulation of oxide production [3,19,73] can be a promising FDA-approved agents arginase 1 and iNOS in tumor macrophages [72]. Thus inhibition of to sensitize cancer cells when combined with other anti-cancer drugs HIF-1α can be a potential therapeutic target against cancer. HIF-1α and therapies. inhibitors such as farnesyl transferase inhibitors and PI3K inhibitors are currently in clinical studies as anti-cancer drugs [19]. Acknowledgement I would like to thank Dr. Kalpit Vora, Vaccine Department. Merck Inc. NJ. Summary and Conclusions USA. for his constructive and intellectual contribution for the preparation of this manuscript. The role of NO in cancer like other biological systems is dependent on the effects and concentration of NO in the microenvironment. Nitric Footnote oxide participates in both direct and indirect reactions in cancer.Nitric Identification and characterization of a particular subset of MDSC with possible oxide may directly interact with its molecular targets suchas metal ions, multiple effector roles in tumorgenesis. Lauren P. Virtuoso, Jamie L. Harden, orit may indirectly react with oxygen and / or superoxide generating Paula Sotomayor, Fuminobu Yoshimura, Nejat K. Egilmez and Mehmet O. Kilinc. Presented at Society for Immunotherapy of Cancer. North Bethesda, November, free nitrogen species which promote nitrosative and / or oxidative 2011. stress [6, 8, 66]. The tumor milieu in most cancers adapts itself to utilize NOto its advantage. Nitric oxide is part of an inflammatory process References [4,12,22,23,65]. Chronic inflammation is associated with several 1. Schreiber RD, Old LJ, Smyth MJ (2011) Cancer immunoediting: integrating cancers including infection-induced cancers [4,9]. 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This article was originally published in a special issue, Mechanisms in Free radical Metabolomics handled by Editor(s). Dr. Eric Kelley, University of Pittsburg, USA

Mechanisms in Free radical J Drug Metab Toxicol Metabolomics ISSN: 2157-7609 JDMT, an open access journal